Design, synthesis, and antiproliferative screening of new quinoline derivatives bearing a cis-vinyl triamide motif as apoptosis activators and EGFR-TK inhibitors

In this work, a congeneric set of quinoline-tethered cis-vinyl triamide hybrids was prepared and evaluated as EGFR tyrosine kinase inhibitors for the management of breast cancer. All of the prepared hybrids were evaluated for their antiproliferative effect against the breast MCF-7 cell line. Among the tested hybrids, compound 6f displayed the most potent antiproliferative activity with an IC50 value of 1.87 μM compared to STU (IC50 = 13.71 μM) as the standard reference. The most promising hybrid, 6f, was found to induce cellular cycle arrest at the G1 phase. Furthermore, the molecular mechanism of this hybrid revealed its ability to induce cellular apoptosis via the mitochondrial-dependent apoptotic pathway. Compound 6f decreased MCF-7 cells' MMP compared to the controls (percentage change value of 57.93%). Further investigation of the selective compound 6f showed that it can inhibit EGFR tyrosine kinase.


Introduction
Cancer remains the leading cause of death in the world, second only to heart disease. 1,2Cancer itself is a dynamic, complex cellular network of uncontrolled growth. 3Of all cancer subtypes, breast cancer is one of the most deadly, and also consistently beckons the largest number of new diagnoses among women. 4,5learly, there is a serious unmet demand for therapies targeting this cancer subtype. 6,7Effective chemotherapeutic treatments with minimal side effects are urgently needed, particularly in the face of the increasing prevalence of drug-resistant tumors. 8erapies based on targeted biological target are intended to be less toxic than conventional chemotherapy. 9,10he epidermal growth factor receptor (EGFR), being one of the most prominent protein kinases, plays a vital role in a series of cellular processes during the life cycle of the cell, such as the regulation of cell migration and cell division. 11,12Multiple prospective clinical trials were initiated to test the efficacy of EGFR-targeted therapy versus standard chemotherapy. 13Clinical trials revealed denite evidence of the superiority of EGFR tyrosine kinase inhibitors over standard chemotherapeutic regimens. 14Targeted inhibitors of EGFR signalling inhibited ligand-induced EGFR auto-phosphorylation and attenuated downstream signalling responsible for proliferation and survival of the cells. 15,16The inhibition of EGFR signalling leads, in most cases, to cell cycle arrest and/or drug-induced apoptosis. 17,18Therefore, EGFR has been regarded as an attractive target for the establishment of chemotherapeutic drugs for many cancers. 19Accordingly, the discovery of new EGFR has attracted a great deal of attention in recent years. 202][23] Several quinoline-based compounds are known to have chemical, biological and therapeutic applications. 24,250][31] The anticancer activity seems to be due to a variety of mechanisms

RSC Advances
PAPER include inhibition of cellular promoting factors such as tubulin polymerization and topoisomerase. 32,33Recently, several research studies have described quinoline derivatives with anticancer efficacy via inhibition of kinases and inhibition of the anti-death Bcl-2 family of proteins 34,35 (Fig. 1).
The amide pharmacophore is an important framework for developing drugs and discovery. 36According to medicinal chemistry sources, one or more amide bonds serve as the basic component in more than 25% of natural and synthetic drugs available in the market. 37The amide functional group enhances the anticancer activity by making molecules more polar as well as forming a hydrogen bond acceptor-donor domain with the target receptor. 38Therefore, the introduction of an amide motif bearing a cis-vinyl group into the quinoline core is likely to signicantly inuence biological activity. 39e aforementioned intriguing ndings, combined with our ongoing quest for more potent anticancer agents, led to the molecular hybridization of the quinoline core and the bioactive triamide motif bearing a cis-vinyl group in order to integrate them into a single molecular framework and achieve a new hybrid that could have potential antiproliferative activity (Fig. 2).The study's goal is to look into the benets of such hybridization in terms of predicted biological activity and to see whether this resulted in better biological activity for the produced hybrids.Lastly, we used in vitro tests to assess the antiproliferative action of the prepared quinoline-cis-vinyl triamide hybrids, as well as FACS and ELISA methods to determine the mechanism of cellular action.Fig. 2 Design strategy adopted for the design of target quinoline tethered cis-vinyl triamides 5 and 6a-h.

Chemistry
The synthetic route used in the preparation of the intermediate and nal quinoline based triamide compounds is depicted in Scheme 1.In the initial step appropriate 2-(4-bromoquinoline)-4-carboxylic acid 2 was obtained from reaction of isatin 1 and 4bromoacetophenone in the presence of 33% KOH in pure ethanol by reuxing which was then heated to reux in pure ethanol in the presence of concentrated sulphuric acid to obtain ethyl quinoline-4-carboxylate 3. 26 This is followed by hydrazinolysis with pure hydrazine hydrate in boiling ethanol to furnish the key intermediate quinoline-4-carbohydrazide compound 4. 40,41 Lastly, nal quinolone-linked triamide derivatives 5 and 6a-h were obtained by the reaction of quinoline-4carbohydrazide 4 and respective methyl 3-aryl-2-(benzamido)-2propenoate derivative in freshly molten sodium acetate and glacial acetic acid by reuxing and yielding 64-78%.The formation of the quinoline linked triamide compounds 5 and 6a-h in general were authenticated by 1 H-NMR, 13 C-NMR and elemental analysis.TLC method.The newly prepared quinoline-triamide molecules' analytical and spectral data ( 1 H-NMR and 13 C-NMR spectra) agreed fully with the suggested structures.

Biology
2.2.1.Antiproliferative activity.The antiproliferative potential of the newly prepared quinoline tethered cis-vinyl triamide derivatives 5 and 6a-h was assessed against the MCF-7 cell line in comparison to Staurosporin (STU) as reference standard drug.Based on obtained in vitro results, the majority of the quinoline tethered cis-vinyl triamide derivatives tested demonstrated moderate to signicant antiproliferative action against the examined cancer cell line.Five compounds; 5, 6b, 6d, 6e and 6f showed 1.06-7.10-foldmore potent antiproliferative activity than the reference STU.Two compounds 6c and 6h showed antiproliferative activity with IC 50 values of 19.25 and 15.63 mM, respectively comparable to STU (IC 50 = 13.28 mM).The rest hybrids 6a and 6g showed modest antiproliferative activity with IC 50 of 59.77 and 30.44 mM, respectively.The antiproliferative activity correlation of quinoline tethered cis-vinyl triamide derivatives 5 and 6a-h showed that among the quinoline-cis vinyl triamide derivatives, compounds bearing electron donating groups such as methyl; 6e (IC 50 = 3.03 mM) and methoxy; 6f (IC 50 = 1.87 mM) showed more potent antiproliferative activity than the other substituted derivatives with electron withdrawing groups and were found to be more potent than STU (IC 50 = 13.28 mM).Another interesting observation is that introducing three groups in the 3,4,5-position of the phenyl ring attached to the cis-vinyl moiety, such as 3,5dibromo-4-acetoxy group; 6g (IC 50 = 30.44mM) and 3,4,5-trimethoxy group; 6h (IC 50 = 15.63 mM), resulted in low antiproliferative activity.It is also notable that replacement of substituted phenyl ring in cis-vinyl moiety with heterocyclic ring such as furan (IC 50 = 6.60 mM) resulted in increase in antiproliferative activity compared to reference drug STU.According to these ndings, the examined quinoline tethered triamide motif bearing cis-vinyl group with furan function or parasubstituted phenyl ring with electron donating group such as methyl or methoxy had a benecial inuence on antiproliferative activity Table 1.

Cell cycle analysis.
The cell cycle is related to a series of events responsible for cell division and duplication. 42The cell cycle presents three distinct phases: G1, S and G2/M phase. 43ue to the importance of the cell cycle in the process of tumor progression, we evaluated if the cell growth inhibition in MCF-7 occurred due to cell cycle arrest using FACS method.In the present study, ow cytometric measurement was utilized to investigate the effect of quinoline tethered  signicantly declined the cellular population at G1 phase.It is observed that the percentage of cells at G1 phase was increased by 1.2-fold compared to controls (Fig. 3).Coordinately, the percentage of cells was decreased at S and G2/M phases from 34.30 and 14.04% in controls to 29.32 and 7.12%, respectively in compound 6f-treated cells.These ndings suggest that quinoline-[2-(4-methoxyphenyl)-cis-vinyl] triamide 6f halted the cell cycle proliferation of the MCF-7 cells at G1 phase.

Apoptosis analysis.
Apoptosis play an important role in organogenesis and in craing complex tissues during embryonic growth, and in the preservation of tissue homeostasis in adult organism. 44Numerous clinical disorders, including cancer, autoimmune illness and infectious diseases, are characterised by the deregulation of apoptosis. 45To ensure the ability of quinoline tethered [2-(4-methoxyphenyl)-cis-vinyl] triamide 6f to activate apoptosis, ow cytometric measurement was carried out using Annexin V which binds to phophatidylserine declared on the outer layed of apoptotic cells and appears uorescent green and PI which stains DNA and penetrates only dead cells.Aer 48 h of treatment with quinoline tethered [2-(4-methoxyphenyl)-cis-vinyl] triamide 6f at the IC 50 concentration (1.87 mM), the percentage of cells that are survived was found to decrease.Additionally, there was a notable rise in the proportion of cells positive for Annexin V.It is notable that, the percentage of primary apoptotic cells was increased from 0.66% in DMSO controls to 9.52% in compound 6f-treated cells.Meanwhile, the proportion of late apoptotic cells rose from 0.21% to 16.65% compared to DMOS controls (Fig. 4).These ndings suggest that quinoline tethered [2-(4methoxyphenyl)-cis-vinyl] triamide 6f provoke apoptosis of MCF-7 cells.
2.2.4.Mitochondrial membrane potential.A major part of apoptosis process is played by mitochondria. 46Several active proteins such as cytochrome C and factor that induces apoptosis are two of the active proteins found in mitochondria. 47The release of such proteins aer inhibition of mitochondrial membrane potential (MMP) is crucial in the propagation of apoptosis. 48To conrm whether quinoline tethered [2-(4-methoxyphenyl)-cis-vinyl] triamide 6f could lower the MMP of tested cancer cell line (MCF-7 cells), ow cytometry was used to track MMP following treatment of the examined cells with 1.87 mM (IC 50 dose value) of compound 6f.Results indicated that the tested cancer cells exhibited signicant decrease in MMP.This decrease in MMP was linked to an  increase in Annexin V positive cells.It is notable that, in comparison to DMSO controls, MCF-7 cells' MMP was 57.93% percent value lower (Fig. 5).According to this, quinoline tethered [2-(4-methoxyphenyl)-cis-vinyl] triamide 6f cause MCF-7 cells to become dysfunction in their mitochondria, which in turn trigger apoptotic cellular death.These outcomes were consistent with early ndings that EGFR-TK inhibitors promote apoptosis via the mitochondrial route.
2.2.5.EGFR tyrosine kinase inhibition analysis.EGFR have been implicated in a variety of cancer indications and other inappropriate mitogenic signalling disorders. 49Clinical evidence demonstrates an association between EGFR overexpression and response to a number of anticancer therapeutics. 50So EGFR and the activated downstream cascades represent a promising target for the establishment of therapeutic agents. 51As result of the effect of quinoline-[2-(4methoxyphenyl)-cis-vinyl] triamide 6f on the G1 phase of the cellular cycle where the protein synthesis required for the cell growth rises, the inhibition of EGFR tyrosine kinase was thought to be the expected mode of cellular action of this hybrid molecule.Thus, the purpose of this experiment was to assess the inhibitory activity of EGFR tyrosine kinase.The quinoline-[2-(4-methoxyphenyl)-cis-vinyl] triamide 6f showed good activity (IC 50 = 0.19 mM), which was comparable to that of Lapatinib (IC 50 = 0.17 mM), a known potent EGFR inhibitor (Fig. 6).So it is concluded that quinoline-[2-(4-methoxyphenyl)-cis-vinyl] triamide 6f exerted its antiproliferative activity through inhibition of EGFR tyrosine kinase.

Conclusions
A new set of quinoline-tethered cis-vinyl triamide hybrids 5 and 6a-h was designed and constructed as inhibitors of EGFR tyrosine kinase for the treatment of breast cancer.All the prepared quinoline-tethered cis-vinyl triamide hybrids were screened for their in vitro antiproliferative activity and revealed moderate to potent activity.Among them, quinoline compounds 6e bearing